Current treatments do not cure individuals of HIV because of HIV's ability to persist as provirus within long-lived immune cells, especially memory T cells. Most attempts to clear these reservoirs have involved the destruction of latently infected cells, and such efforts to date have been unsuccessful. A more elegant solution to the problem of HIV infection would be the ability to eliminate or otherwise disable integrated HIV without inducing death of infected cells. We have recently begun to evaluate a novel approach to achieve this, via the use of novel, highly specific enzymes known as homing endonucleases (HEs). HEs are proteins that specifically target long (~20 base pair) sequences in double stranded DNA. The long recognition sites ensure high specificity and prevent off-target effects. Upon recognition of their target sites, HEs induce DNA double strand breaks (dsb) at the sites, which in mammalian cells are most commonly repaired via non-homologous end joining (NHEJ). NHEJ repair is error prone, and can lead to mutation typically resulting in deletions surrounding the cleavage site ranging from 1 base to several kilobases. In our preliminary studies, we created an engineered reporter virus containing the recognition site for the natural HE l-Anil, to demonstrate that integrated provirus can be successfully targeted for attack, cleavage, and mutagenesis by HEs. Based on this success, we have now assembled a team of investigators with the complementary expertise necessary to rationally redesign natural homing endonucleases such that they will recognize conserved sequences within SHIV. Our aims are as follows. 1) Design variant homing endonucleases targeting integrated SHIV sequences. We will build upon existing collaborations with Drs. David Baker and Andrew Scharenberg, with whom we have already used computational and bacterial selection approaches to design novel HEs recognizing conserved sequences from HIV. 2) Optimize the DNA-modifying efficacy of SHIV-specific homing endonucleases. We will do this by directed evolution processes for selection of increased activity, and by linking the HEs to TREX2, which we have shown increases the incidence of targeted mutation. 3) Establish the optimal methods for delivery of SHIV-specific HEs to macaque CD4+ T cells and CD34+ stem cells, and determine the ability of HEs to clear viable provirus from these reservoirs. During autologous hematopoietic stem cell transplantation in HIV-infected individuals, between 10[5] and 10[10] copies of HIV provirus are re-infused along with the graft, and the ability to purge viable virus from these cells before reintroduction may be of clinical benefit to the patient.